Tuberculosis continues to cause tremendous morbidity and mortality throughout the world's population. Critical in establishment of a M. tuberculosis (M.tb) infection are entry and survival in the macrophage. The M.tb cell envelope is heavily mannosylated with the abundant lipoglycans lipoarabinomannan (ManLAM), lipomannan (LM), and phosphatidyl inositol mannosides (PIMs). These lipoglycans bind to C-type lectins expressed on macrophages and dendritic cells (DC). The mannose receptor (MR) is a C-type lectin that mediates phagocytosis of virulent strains of M.tb by human macrophages. DC-SIGN is expressed abundantly on DCs and is also expressed on alternatively activated macrophages such as alveolar macrophages. ManLAM is a major ligand for the MR and DC-SIGN. We hypothesize that the nature of surface mannosylation of M.tb has a major impact on the ability of the bacterium to interact with C-type lectins (the MR and DC-SIGN) and thereby modulate macrophage function and host responses. The cytosolic nucleotide mannose carrier GPD-mannose is an essential donor either directly or indirectly in the biosynthesis of LAM, LM, and PIMs as well as cell wall mannoproteins. This proposal focuses on the core enzymes that are required to produce GPD-mannose in mycobacteria. Genes encoding these enzymes will be over-expressed or knocked out inM. smegmatis and M.tb and mutant bacteria with altered surface mannosylation will then be studied in human macrophages and in the mouse aerosol model of TB pathogenesis. One such gene is M.tb manB which we have found encodes a functional phosphomannomutase. Overexpression of manB in M. smegmatis leads to a significant increase in LAM, LM, and PIMs produced as well as a 13-fold increase in bacterial association with macrophages. In addition to the targeted gene approach, we will screen M.tb libraries for clones that are altered in surface mannosylation. Specific aims: Aim 1: Clone, over-express and knock out a defined set of M.tb genes involved in GDP-mannose biosynthesis, Aim 2: Determine whether mycobacteria mutants are altered in surface mannosylation, Aim 3: Determine how genes involved in mannose biosynthesis impact M.tb interactions with the MR and DC-SIGN, the biology of bacteria in human macrophages, and M.tb pathogenesis in the mouse, Aim 4: Screen a M.tb transposon library and a M. smegmatis library over-expressing M.tb genes using Salmonella mannose-binding (type 1)pili and surfactant protein D to identify mutants and clones altered in surface mannosylation. The overall goal of this proposal is to combine molecular, biochemical, and cell biology techniques to identify key enzymes for GDP-mannose biosynthesis in M.tb and, by altering then- level of expression, determine their impact on 1)M.tb cell wall mannosylation and 2) the biology of bacterial behavior in human macrophages and the mouse. Information gained from these studies should enhance our knowledge of TB pathogenesis and also potentially identify new therapeutic targets since mannose metabolism has been shown to be essential for the survival of mycobacteria.